Optimal brain function relies upon the delicate equilibrium between excitatory and inhibitory neurotransmission. A slight disturbance in this equilibrium can trigger significant neurological disorders. In the mature brain, glycine assumes a dual responsibility by not only coordinating inhibitory processes through the activation of classical pentameric glycine receptors (pGlyRs), but also contributing to excitatory neurotransmission in its capacity as a co-agonist of N-methyl-D-aspartate receptors (NMDARs). The functional impact of glycine through pGlyRs has been extensively scrutinised, particularly in the brainstem and spinal cord. By contrast, the role and functions of glycine within the forebrain remain enigmatic and several fundamental questions persist. Recently, two novel classes of glycinergic receptors, excitatory (eGlyRs) and metabotropic (mGlyRs) have been unveiled, necessitating a paradigm shift in our understanding of glycine neurotransmission within the brain. Combining a variety of approaches in mouse brain, we provide evidence that cholinergic interneurons (ChINs) in the striatum express all three types of glycine receptors. We find that glycine strongly regulates ChIN activity and that ambient striatal levels of glycine operate a tonic excitatory influence via at least eGlyRs. Furthermore, we find that activation of striatal eGlyRs can modulate dopamine release downstream of ChIN activation. Collectively, our results unveil significant function of glycine in the striatal circuit, particularly in its influential modulation of ChINs, placing them as key integrators of glycinergic neurotransmission. These findings provide compelling support for a more expansive role of glycine in forebrain neurotransmission.